Mechanotransduction - the cellular response to mechanical stress - is thought to occur in the cytoplasm and is vital to many fundamental cell functions. However, how applied stresses are propagated within the cytoplasm and transduced into cellular responses is unknown. In this application we propose to map load-induced displacements and stresses in the cytoskeleton, the putative stress-bearing network in the cytoplasm. Preliminary data establish that we can track intracellular cytoskeletal structures marked with green fluorescent protein using a synchronous detection method. We can also measure the spatial distribution of displacements of these structures and compute intracellular stresses that arise in response to a small localized mechanical deformation imposed on the cell from the outside. We were surprised to find that the induced fields of intracellular strain and stress did not decay rapidly in space, as would be predicted from all current models of cell mechanics, but rather exhibited focused stress propagation over long distances. Here we propose four Specific Aims: Aim 1 is to further develop the technology to quantify intracellular displacement and stress fields in three dimensions in the cytoskeleton. Aim 2 is to test the hypothesis that the prestress mediates long distance stress propagation and to identify the origin of stress and strain concentration in the cytoskeleton. Aim 3 is to map the dynamic features of the cytoskeletal structures in three dimensions in response to localized oscillatory loads. Aim 4 is to determine the roles of vimentin, cytoskeletal crosslinking protein plectin, and focal adhesion proteins vinculin and talin in cytoskeletal stress propagation. The proposed bioengineering research combines novel mechanical measurements of the contractile state with mathematical analysis of cell deformation. The experimental method is to measure with high spatial and temporal resolution the intracellular deformation field in response to localized mechanical loading, and to characterize the mechanical state and cytoskeletal structure during specific interventions. The current poject may have implications in elucidating specific loci and structural pathways for mechanotranduction at sites deep in the cytoplasm.